This method is after Marchuk,D.,et al.,1991,Nucl.Acids Res.19(5),pp1154. You will need: 1) Digest plasmid to be used with a blunt-end restriction enzyme (I use EcoRV and pBluescript SK-). 2) Clean digestion reaction by phenol/chloroform extraction and ethanol precipitation. 3) Redissolve DNA in 1 x Taq PCR buffer (I use Taq and buffer from Promega but I think any non-proofreading Taq will work) to give approx. 1ug DNA/20 ul. 4) Add dTTP to a final conc. of 2mM (typically from a 100mM stock solution). 5) Add Taq polymerase to give 1 unit/ug DNA/20ul. 6) Incubate mixture at 72°C for 2 hours. 7) Extract mixture once with phenol/chloroform and once with chloroform. 8) Ethanol precipitate DNA,wash with 70% ethanol,dry and redissolve in TE at an approx. conc. of 25ng/ul. 9) Quantitate DNA (I use an approx.quantitation by gel fluorescence against standards). 10) For ligations,use approx.25-50ng T-vector and sufficient PCR product to give a 3:1,insert:vector molar ratio(I use T4 Ligase from Gibco-BRL together with their PEG-based buffer). 11) Ligate O/N at 15°C. 12) Transform 1/5 ligation mix into competent E.coli(I use DH5a cells at approx.5 x 107 cfu/ug pUC19). |
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